The senomorphic impact of astaxanthin on irradiated rat spleen: STING, TLR4 and mTOR contributed pathway

Abstract

Objectives: Exposure of spleen tissues to ionizing radiation during radiotherapy can induce cellular stress and immune-dysfunction leading to cellular senescence.

Introduction: The process of a cancerous development is facilitated by the accumulation of senescent cells. This justifies the incorporation of anti-senescent medications during splenic irradiation (SI).

Methods: In this study senescence was induced in the spleen of male albino rats by radiation exposure (5Gy-single whole body gamma-irradiation) then after 2 weeks, oral astaxanthin regimen was started once daily in a dose of 25 mg/kg for 7 consecutive days. Concurrent control groups were carried out.

Results: the present data reflected that irradiation provoked an increase in the oxidative stress biomarkers (nitric oxide, lipid peroxidation and total reactive oxygen species levels)and the inflammatory biomarkers (Myeloperoxidase and interleukin-6). In addition irradiation led to the over expression of stimulator of interferon genes (cGAS-STING), mammalian target of rapamycin (mTOR) and Toll-like receptor 4 (TLR4) along with the lactate dehydrogenase (LDH), cyclin-dependent kinase inhibitor 1 (p21) cyclin-dependent kinase inhibitor 2A (p16) increment with elevation of tumor suppressor protein (p53) level. However, reduced glutathione contents and catalase activity were reduced post irradiation in spleen tissues, all these changes reflecting induction of cellular senescence. Astaxanthin treatment showed an improvement in the antioxidant/oxidative stress balance, inflammatory biomarkers, histopathological examination and immunohistochemical expressions of the tested proteins in the irradiated rats.

Conclusion: the current findings offer a new insight into the senomorphic effect of astaxanthin following radiation-induced spleen senescence via STING, mTOR, and TLR4 signalling pathways.

Keywords: astaxanthin; cellular senescence; mammalian target of rapamycin; stimulator of interferon genes; toll-like receptor 4.

Figure 1.

Astaxanthin ameliorates oxidative stress in spleen tissues of irradiated rats in senescence-induced model. A: GSH, B: Catalase, C: TBARS, D: NOx. Values are given as mean ± S.E, n = 6 statistically differences were assessed by one –way ANOVA followed by Tukey-Kramer multiple comparison test.*: significant change from control at p < .05, #: significant change from Sc group at p < .05.

Figure 2.

Astaxanthin ameliorates inflammation induced by irradiation in spleen in senescence-induced model. A: MPO, B: IL6, C: IL6 expression, D: photomicrographs of immune-stained spleen sections IL-6; showing negative expression of IL-6 , in control and ASX group (a&b), diffuse intense expression in irradiated group (c), and significant decreased expression of IL-6 in ASX treated group (d). Image analysis of the optical density of the positive brown color. Values are given as mean ± S.E, statistically differences were assessed by one –way ANOVA followed by Tukey-Kramer multiple comparison test.*: significant change from control at p < .05, #: significant change from Sc at p < .05.

Figure 3.

Astaxanthin protects against cellular integrity damage and senescence markers in spleen of irradiated rats. A: LDH, B: P21, C: STING relative gene expression, D: p16, E:P53. Values are given as mean ± S.E, n = 6 statistically differences were assessed by one –way ANOVA followed by Tukey-Kramer multiple comparison test.*: significant change from control at p < .05, #: significant change from Sc at p < .05.

Figure 4.

Astaxanthin improves the histological alterations in the splenic tissue in a rat-model of radiation-induced senescence . Photomicrographs of spleen sections stained with H&E.(a and b) Control and ASX-administrated groups; showing normal histologic structure, normal white pulp follicles (Fo), periarteriolsheeth (PA), mantel zone (MZ), and red pulp (RP). (c-f) SC group showing (c) marked white pulp follicles atrophy and lymphocytic depletion (square), (d) many tangible body macrophages (arrow and insert) and lymphocytolysis in the follicles and in (e) the periarteriolar area (PA), with the reticular mash underneath appearance (insert). The ASX treated group exhibited a significant recovery in the size and intensity of the mantel zone (MZ) of white pulp follicles, along with a modest to moderate degree of depletion of follicular (F) lymphocytes and appearance of a few tingible body macrophages (arrow).

Figure 5.

Astaxanthin administration down regulates the splenic mTOR (A) and TLR4 (B) expression splenic tissue in radiation induced senescence model (immunohistochemical study). (C) Photomicrographs of immune-stained spleen sections for TLR4 and mTOR; showing negative expression of TLR4 and mild expression of mTOR in control and ASX groups, diffuse intense expression in irradiated group, and significant decreased expression of the two markers proteins in ASX treated groups, Image analysis of the optical density of the positive brown color. Values are expressed as mean ± SE. Data were analyzed by using one –way ANOVA followed by Tukey-Kramer multiple comparison test.*: significant change from control at p < .05, #: significant change from Sc at p < .05.